1. Field of the Invention
The invention provides new methods for synthesis of nucleotide-based compounds and libraries of such compounds. Compounds of the invention are useful for a variety of therapeutic applications, including treatment of viral or bacterial infections and associated diseases and disorders.
2. Background
The important initial step in the development of therapeutic agents is the discovery of compounds that bind to a protein, enzyme or receptor of interest. Through careful structure/activity work of resulting active compounds, one arrives at a lead compound for further development into a clinical candidate. This traditional process of drug discovery is a long and arduous endeavor. Often it takes 10 to 15 years before a new drug makes it into the marketplace.
Recent advances in molecular biology and genomics have led to identification of new molecular targets for drug discovery. As a result of the limitation of traditional drug discovery, new approaches to the discovery of therapeutics have been developed. In the more modern approaches, large libraries of diverse compounds are synthesized by a number of methods and subjected to high throughput in vitro screening against a particular molecular target implicated in a disease. The active compounds so identified are then subjected to Structure-Activity Relationship (SAR) work to eventually identify the lead compound.
Modern drug discovery approaches entail the synthesis and screening of libraries of compounds. The design and synthesis of such libraries is often based on a unique molecular skeleton or scaffold. By incorporating a variety of structural elements into a scaffold, local as well as global molecular diversity can be achieved which facilitates specific interactions between a ligand and its receptor. The structural elements contribute to molecular diversity by variable spatial display of ionic, hydrogen-bonding, charge-transfer and van der Waals interactions thus allowing for the selection of the best xe2x80x98fitxe2x80x99 between the ligand and its receptor.
Traditionally, libraries have been constructed using solid-support synthesis methods, such as synthesis of a library on xe2x80x98beadsxe2x80x99. Solid support methods are useful because reactive products can be readily isolated in a relatively pure form by simply washing away excess reagents and solvents from the support matrix, something that is not possible with solution based methods.
One method for generating compound libraries utilizes a discrete compound approach. In the discrete compound approach, compounds are synthesized in parallel each in a separate reaction vessel. The identity of each compound is known or can be ascertained by analytical methods. Various methods for constructing discrete compound libraries are known in the art. For example, the Pin method (H. M. Geyson et al., PNAS, USA 81: 3998-4002 (1984)) utilizes polyethylene pins placed in a 96-well supporting block. Each pin is coated with polymeric material that is derivatized for anchoring functional groups. The reactions can be run on 100 nmol to 50 micromol scale and the products subjected to multiple biological assays. The Diversomer apparatus approach (S. H. Dewitt et al., PNAS, USA 90: 6909-6913 (1993)) utilizes a series of porous gas dispersion tubes which serve as containers for resin beads and reagents and solvents are placed in vials mounted on a reservoir block. The ends of the gas dispersion tubes are placed in the vials and the reagents are allowed to diffuse through the porous membrane and contact the resin support. The apparatus can be placed in a manifold with an injectable gasket. The porous frit apparatus utilizes each well of a deep well microtiter plate fitted with porous frits. The plate is clamped on to a viton gasket. In between synthetic steps in a sequence, the reaction solution can be drained and the resin rinsed by removal of the viton gasket. The spatially addressable, light directed parallel synthesis method utilizes a photolithographic method to synthesize 100,000 separate compounds. The synthesis is done on a silicon wafer (chip) that is functionalized to attach to a leader molecule which carries a photolabile protecting group at its reaction site. Once unmasked by illumination, the reactive group is unmasked which can then enter into a specific chemical reaction with a reactant. The library of compounds remain tethered to the solid support. The structure of the compound in each specific location is known.
Another method for generating compound libraries utilizes a mix and pool synthesis approach. This approach allows large libraries of compounds to be synthesized by pooling different sets of support-bound intermediates. However, this method only works when all of the reactants in a mixture have similar reactivities. Reaction conditions need to be optimized before attempting a split and pool strategy. This strategy has been used to synthesize libraries of peptides and oligonucleotides. Various mix and pool synthesis approaches are known in the art. For example, Houghten et al. (C. Pinilla et al., Biopolymers, Pept. Sci., 37: 221-240 (1995), pioneered this approach by preparing pools of compounds that each contain structurally defined building blocks at one or two positions. Once the pool with the highest activity is identified in an in vitro assay, the deconvolution process begins. Iterative rounds of synthesis and biological assays are carried out until a molecule with the highest activity is identified. Modifications of this approach include the positional scanning approach developed by Houghten et al. (C. Pinilla et al., Biopolymers, Pept. Sci., 37: 221-240 (1995) and the orthogonal approach developed by Tartar et al. (B. Deprez, et al., J. Am. Chem. Soc., 117: 5405-5406 (1995). Another mix and pool synthesis approach utilizes beads encoded by oligonucleotides of known sequence to trace compounds.
Biological assays used to test the activity of compound libraries can be carried out with the compounds immobilized on a solid support or in solution. For example, a resin-bound library can be treated with a fluorescent-labeled receptor and the compound-bound receptors isolated using a fluorescence activated cell sorting instrument. Structure determination can be done, for example, by sequencing or mass spectrometry analysis. When assays are performed in solution, the compounds need to be released from the solid support. A portion of the beads are released and contacted with the receptor. The active compounds are then traced back to the original bead. Structure determination can be performed by analytical methods.
See also: C. Pinilla et al., Biopolymers, Pept. Sci 37: 221-240 (1995); S. H. DeWitt et al., PNAS, USA 90: 6909-6913 (1993); B. Deprez et al., J Am. Chem. Soc. 117: 5405-5406 (1995); H. M. Geyson, et al. PNAS, USA 81: 3998-4002 (1984); G. Jung et al., Angew. Chem. Intl. Ed. Engl. 31: 367-383 (1992); M. R. Pavia, et al. Bioorg. Med. Chem. Lett. 3: 387-396 (1993); E. M. Gordon et al., J. Med. Chem. 37: 1385-1401 (1994); L. A. Thompson et al., Chem. Rev. 96: 555-600 (1996); S. Verma et al., Annu. Rev. Biochem. 67: 99-134 (1998); S. L. Beaueage et al., Tetrahedron Lett. 22: 1859-1862 (1981); R. P. Iyer et al., In Comprehensive Natural Products, D. H. R. Barton and K. Nakanishi Eds., Elsevier Science. Vol 7 (In press); A. D. Barone et al., Nucl. Acids Res. 12: 4051-4061 (1984); R. P. Iyer et al., J. Am. Chem. Soc. 112: 1253-54 (1990).
We have now found new nucleotide-based compounds that are useful for a variety of therapeutic applications, including to treat against viral or bacterial infections.
The invention also provides new methods for synthesis of nucleotide-based compounds and new libraries of such compounds. In particular, the invention provides new methods for construction of compound libraries utilizing a nucleic acid-based (NAB) scaffold. This approach enables incorporating structural elements that can provide both xe2x80x9csequence-specificxe2x80x9d interactions (e.g., hydrogen-bonding interactions between nucleobases) as well as xe2x80x9cshape-specificxe2x80x9d motifs (e.g., bulges and stem-loop structures) that can allow specific recognition of other nucleic acids and proteins. Libraries based on NAB scaffold can potentially mimic the molecular recognition that exists between cellular macromolecules and biomolecules such as hormones, nucleotides and their receptors.
The invention provides methods for constructing compound libraries by solution-phase or solid-phase approaches. Preferred library syntheses of the invention are carried out on a solid support. Suitable solid supports include, for example, pins, beads, resins, chips, etc.
Preferred library syntheses of the invention include use of columns capable of agitation (e.g. spin or other rotation) and that may suitably contain a resin support material. Reactants are placed in the column, and the column preferably shaken or otherwise agitated during reaction. Additional reactants can be added to provide repeated reaction cycles. Reagents and reaction products also can be conveniently separated and removed from the column, e.g. by centrifuging a reaction column to facilitate removal (e.g. by filtration) of desired material.
Other aspects of the invention are disclosed infra.